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This pathway also protects against neurodegenerative diseases.1,2 The hallmarks of amyotrophic lateral sclerosis, Parkinson's disease, Lewy-body dementia, Huntington's disease, and Alzheimer's disease vary, but all are characterized by the presence of abnormal proteins in intracellular inclusions that are generally associated with ubiquitin and proteasomes.2 These diseases seem to result from the accumulation of a toxic protein species, although there is increasing evidence that these amyloid inclusions are a correlate rather than the immediate cause of the disease2,3 and that the neuronal pathology results from smaller soluble microaggregates of the aberrant protein.
The mechanism by which these amyloid-like proteins interfere with normal function and eventually promote apoptosis remains uncertain. One often-proposed hypothesis is that the ubiquitin–proteasome pathway normally eliminates these misfolded toxic proteins, but they gradually accumulate, and eventually, especially in aged persons, overwhelm the capacity of this proteolytic system and interfere with its functioning. The further buildup of the misfolded proteins finally causes a failure of the proteolytic mechanisms of neurons that are necessary for normal cell function and survival.2 Direct evidence that the abnormal toxic proteins in these various diseases can inhibit the functioning of the proteasome pathway has been lacking.
Recently, however, Kristiansen et al.3 presented strong evidence that soluble aggregates of the toxic protein may cause prion disease by specifically inhibiting the function of the 26S proteasome. Prion diseases are a set of transmissible, fatal neurodegenerative diseases associated with spongiform encephalopathy, including the diseases that occur in humans, Creutzfeldt–Jakob disease, the Gerstmann–Sträussler–Scheinker syndrome, and kuru; in sheep, scrapie; and in cows, bovine spongiform encephalopathy (or "mad cow disease").4 More than 150 people in Britain have died of a variant of Creutzfeldt–Jakob disease acquired through ingestion of prion-containing beef. These diseases had long represented a biologic mystery, primarily because the transmissible agent was shown to lack nucleic acids, but the seminal studies of Stanley Prusiner and Charles Weissmann established that the diseases are caused by a toxic form of the prion protein. This protein is able to enter cells and induce a conformational rearrangement of a normal soluble, monomeric cell constituent, the prion protein (PrPC), into a toxic transmissible species (PrPSc).4 In this pathogenic conformation, the prion is primarily insoluble, is resistant to proteases or detergents, has more amyloid-forming β-sheet structure, and forms larger aggregates than the normal species. This conformational change and the resulting accumulation of PrPSc ultimately cause severe loss of neurons, gliosis, and spongiform appearance. Accordingly, mice that do not express the normal protein are not susceptible to the disease.3,4
A major gap in our understanding has been how the conversion of PrPC to PrPSc eventually kills neurons. To clarify the mechanism of neurotoxicity, Kristiansen et al. tested whether PrPSc interferes with proteasome function, using in vivo and in vitro approaches. In the ubiquitin–proteasome pathway, proteins are targeted for rapid degradation by covalent linkage to a chain of ubiquitin molecules, which marks them for rapid hydrolysis by the large 26S proteasome complex. When tagged with a ubiquitin chain, substrates bind to the proteasome's 19S regulatory component, which disassembles the ubiquitin chain and recycles the ubiquitin molecules. Then through a multistep ATP-dependent process that is only now beginning to be understood, the 19S particle's ATPases unfold the substrate and translocate it through a narrow, gated entry channel into the 20S proteasome for degradation (Figure 1).5 Inside this hollow cylindrical particle, the doomed protein is cleaved to small peptides, which are then released from the proteasome and rapidly degraded to amino acids by cytosolic peptidases.
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By infecting transgenic mice expressing this reporter protein with prions, Kristiansen et al. showed that PrPSc induced similar defects in the brain's capacity to degrade this fluorescent protein. In infected neurons of these mice, ubiquitinated deposits accumulated in the cytosol, reminiscent of those found in various adult-onset neurodegenerative diseases, and in cells treated with pharmacologic inhibitors of the proteasome.3 Although the decreased degradative capacity is consistent with a loss of proteasomal activity, it could also be due to defects in ubiquitination or other steps in the pathway. However, the authors obtained further supporting data using purified 26S proteasomes and PrPSc isolated from infected mouse brain, recombinant mouse PrP folded into an 
-helical structure resembling the native PrPC, or a predominantly β-sheet species that mimics the properties of PrPSc. A clear reduction in the activity of the purified proteasomes (as well as those in neurons) occurred only with the aggregated forms of PrPSc. This inhibition was shown to result from a stoichiometric association of the oligomeric species with the proteasomes.
Although it is now clear that cytosolic PrPSc can interfere with the proteasome's peptidase activities and that infected neurons are defective in protein degradation, there is no proof that this is the only toxic mechanism, or even the critical one leading to neuronal loss in prion diseases. Loss of proteasomal function affects many critical cellular processes and can induce apoptosis. In addition to serving as a quality-control system, this pathway is critical in regulating cell signaling networks and metabolic pathways and achieves exquisite selectivity because of the presence in humans of hundreds of different ubiquitin ligases that function in the removal of different proteins.1 Not surprisingly, in cellular models of all neurodegenerative diseases, treatment with inhibitors of the proteasome promotes pathogenesis and neuronal death. The proteolytic sites of the 20S proteasome are the target of the anticancer drug bortezomib (Velcade), which is widely used in the treatment of multiple myeloma — a cancer that seems to be particularly dependent on proteasomes for survival and for the degradation of misfolded immunoglobulins. An important feature of this drug's selectivity is that it does not enter the central nervous system, where it would probably accelerate the onset of neurodegenerative disease.
It also remains unclear exactly how small aggregates of the PrPSc inhibit the cleavage of peptides by the proteasome. The "decision" of which proteins are translocated into the particle and degraded depends on the ring of ATPases in the 19S regulatory component, which is probably the site where PrPSc oligomers bind (Figure 1). Normally, cell proteins that are destined for degradation bind to and are unfolded by these ATPases before being translocated into the 20S particle. However, the exceptional stability of PrPSc makes it unlikely that these structures can be disassembled similarly, and the microaggregates of PrPSc are probably too large to traverse the ATPase ring and the narrower, gated pore into the 20S particle. Thus, they may behave like a sticky cork that interferes with the entry of other substrates into the 20S particle. Such a simple mechanism is now amenable to testing.
Source Information
From the Department of Cell Biology, Harvard Medical School, Boston.
References
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